Methods for determining and achieving therapeutically effective doses of anti-cd47 agents in treatment of cancer

ABSTRACT

Methods are provided for treating a subject with an anti-CD47 agent.

CROSS REFERENCE

This application is a Continuation and claims benefit of U.S.application Ser. No. 17/930,621, filed Sep. 8, 2022, which claims thebenefit of U.S. application Ser. No. 16/089,115, filed Sep. 27, 2018,now U.S. Pat. No. 11,472,878, issued Oct. 18, 2022, which claims thebenefit of PCT Application No. PCT/US2017/027662, filed Apr. 14, 2017,which claims benefit of U.S. Provisional Patent Application No.62/323,330 filed Apr. 15, 2016, and U.S. Provisional Patent ApplicationNo. 62/427,679, filed Nov. 29, 2016, which applications are incorporatedherein by reference in their entirety.

BACKGROUND

The large majority of cancers worldwide are solid tumors. In 2016, it isestimated that over 1,600,000 people will be newly diagnosed with amalignant solid tumor in the US (Siegel et al. (2016), Cancerstatistics, 2016. CA: A Cancer Journal for Clinicians, 66:7-30). Currentstandards of care for solid tumors include surgical excision,radiotherapy, cytotoxic chemotherapy, and molecularly targeted smallmolecules and monoclonal antibodies (mAbs). Despite these therapies,most patients with metastatic cancer will die of the disease and/ortreatment complications. Small molecules that target cancers havelimited efficacy as single agents because of pre-existing or emergentresistance and usually exhibit toxicity to normal cells.

The development of therapeutic mAbs has substantially impacted treatmentof some types of cancer. Conventionally these recombinant proteinsspecifically bind cancer cells and either block signaling pathways ormark them for destruction by the immune system. However, targeted mAbsexist for only a few cancers, even the most effective mAbs may requirecombination therapy with conventional chemotherapy, and often produce anincomplete therapeutic response. In many patients, the disease becomesresistant to mAb treatment by loss of the antibody target (when themolecule is not essential for tumor cell survival) or by developingresistance to tumor killing. Usually patients experience a relapse oftheir disease.

CD47 has been identified as a key molecule mediating cancer cell evasionof phagocytosis by the innate immune system. CD47 appears to be anindispensable means by which cancer cells, including cancer stem cells,overcome intrinsic expression of their prophagocytic, “eat me,” signals.The progression from normal cell to cancer cell involves changes ingenes and/or gene expression that trigger programmed cell death (PCD)and programmed cell removal (PCR). Many of the steps in cancerprogression subvert the multiple mechanisms of PCD, and the expressionof the dominant antiphagocytic signal, CD47, may represent an importantcheckpoint.

CD47 expression is increased on the surface of cancer cells from a largenumber of diverse human tumor types including the following primarymalignancies: head and neck, melanoma, breast, lung, ovarian,pancreatic, colon, bladder, prostate, leiomyosarcoma, glioblastoma,medulloblastoma, oligodendroglioma, glioma, lymphoma, leukemia, andmultiple myeloma. In murine xenograft studies, it has been shown thatCD47-blocking antibodies inhibit human cancer growth and metastasis byenabling the phagocytosis and elimination of cancer stem cells andcancer cells from various hematologic malignancies and several solidtumors.

CD47 serves as the ligand for SIRPα, which is expressed on phagocyticcells including macrophages and dendritic cells. When SIRPα is activatedby CD47 binding, it initiates a signal transduction cascade resulting ininhibition of phagocytosis. In this way, CD47 functions as anantiphagocytic signal by delivering a dominant inhibitory signal tophagocytic cells. It has been demonstrated that a blocking anti-CD47 mAbenabled the phagocytic elimination of cancer stem cells and cancercells.

In mouse xenografts, CD47-blocking mAbs inhibit human xenograft tumorgrowth and metastasis by enabling the phagocytosis and elimination ofcancer cells from various hematologic malignancies and solid tumors.Furthermore, CD47 blocking mAbs synergize with the established cancercell targeting mAbs rituximab, trastuzumab, and cetuximab to enhancetherapeutic efficacy in some tumor types.

Methods for effective delivery of antibodies that block CD47 interactionwith SIRPα are of clinical interest, and are provided herein.

SUMMARY OF THE INVENTION

Methods are provided for treating an individual with a therapeutic doseof anti-CD47 agent. The methods of the invention administer effectivepriming and therapeutic doses of an agent that binds to CD47, which ispresent on cancer cells and which can be present on red blood cells(RBC). An anti-CD47 agent for use in the methods of the inventioninterferes with binding between CD47 present on a cancer cell to SIRPαpresent on a phagocytic cell. Generally both such cells are present inthe individual being treated. Such methods, in the presence of apro-phagocytic signal, can increase phagocytosis of the target cancercell while reducing undesirable side effects on RBC populations.

The subject methods can be used to treat a subject for cancer with anagent that binds to CD47, including anti-CD47 antibodies, where the termantibodies encompasses antibody fragments and variants thereof, andSIRPα polypeptides, e.g, multivalent polypeptides comprising a SIRPαsequence. Suitable agents include, without limitation, Hu5F9, includingHu5F9-G4; CC-9002; TTI-621 and bivalent, tetravalent, etc. high affinitySIRPα polypeptides.

As has been previously described, a therapeutic dose of a CD47 bindingagent can lead to a loss of erythrocytes (RBCs) and anemia.Administration of a priming dose of CD47 binding agent significantlyreduces toxicity due to loss of older erythrocytes while sparing youngererythrocytes. Without being bound by theory, it is believed that theprimer agent increases production of reticulocytes (younger RBC), whichmay be more resistant to CD47 mediated phagocytosis and therefore areless susceptible to loss during subsequent administration of theanti-CD47 agent. Methods are provided for determining an appropriatetiming dose for preclinical or clinical use, by determining the receptoroccupancy of CD47 of blood cells, e.g. RBC and WBC. It is shown hereinthat a suitable priming dose provides for greater than about 50%receptor occupancy on RBC. The methods of determining the priming dosecan be applied to any CD47 binding agent.

In some embodiments of the invention, an effective priming dose ofHu-5F9G4 is provided, where the effective priming dose for a human isaround about 1 mg/kg, e.g. from at least about 0.5 mg/kg up to not morethan about 5 mg/kg; from at least about 0.75 mg/kg up to not more thanabout 1.25 mg/kg; from at least about 0.95 mg/kg up to not more thanabout 1.05 mg/kg; and may be around about 1 mg/kg.

An initial dose of a CD47 binding agent, including but not limited to apriming dose, may also lead to hemagglutination for a period of timeimmediately following infusion. Without being bound by the theory, it isbelieved that the initial dose of a multivalent CD47 binding agent maycause cross-linking of RBC bound to the agent. In certain embodiments ofthe invention, a CD47 binding agent is infused to a patient in aninitial dose, and optionally in subsequent doses, over a period of timeand/or concentration that reduces the possibility of hematologicmicroenvironments where there is a high local concentration of RBC andthe agent.

In some embodiments of the invention, an initial dose of a CD47 bindingagent is infused over a period of at least about 2 hours, at least about2.5 hours, at least about 3 hours, at least about 3.5 hours, at leastabout 4 hours, at least about 4.5 hours, at least about 5 hours, atleast about 6 hours or more. In some embodiments an initial dose isinfused over a period of time from about 2.5 hours to about 6 hours; forexample from about 3 hours to about 4 hours. In some such embodiments,the dose of agent in the infusate is from about 0.05 mg/ml to about 0.5mg/ml; for example from about 0.1 mg/ml to about 0.25 mg/ml.

In some embodiments a priming dose is fractionated into two or moresubdoses, delivered over a period of time from about 1 day, about 2days, about 3 days, about 4 days, about 1 week, about 10 days, about 2weeks.

In other embodiments, an initial dose of a CD47 binding agent, e.g. apriming dose, is administered by continuous fusion, e.g. as an osmoticpump, delivery patch, etc., where the dose is administered over a periodof at least about 6 hours, at least about 12 hours, at least about 24hours, at least about 2 days, at least about 3 days.

In some embodiments a priming dose may be delivered through asub-cutaneous route, by injection, patch, osmotic pump, and the like asknown in the art.

Following administration of the priming agent, and allowing a period oftime effective for an increase in reticulocyte production, a therapeuticdose of an anti-CD47 agent is administered. The therapeutic dose can beadministered in number of different ways. In some embodiments, two ormore therapeutically effective doses are administered after a primeragent is administered, e.g. in a weekly dosing schedule. In someembodiments a therapeutically effective dose of an anti-CD47 agent isadministered as two or more doses of escalating concentration, in othersthe doses are equivalent.

In some embodiments of the invention, the therapeutic (maintenance) doseis sufficient to achieve a circulating level of greater than 100 μg/mlfor a sustained period of time. In some such embodiments the anti-CD47agent is the antibody 5F9. In some embodiments the sustained period oftime is up to about 1 week. In some embodiments the sustained period oftime is up to about 10 days. In some embodiments the sustained period oftime is up to about 2 weeks. In some embodiments the maintenance dose isfrom about 10 mg/kg to about 25 mg/ml, from about 12.5 mg/kg to about22.5 mg/kg, from about 15 mg/kg to about 20 mg/kg, from about 17.5 mg/kgto about 20 mg/kg, from about 10 mg/kg to about 20 mg/kg. Themaintenance dose may be administered at a periodicity that provides forsustained serum levels of greater than about 100 μg/ml, whereadministration may be weekly, every 8 days, every 9 days, every 10 days,every 11 days, every 12 days, every 13 days, every two weeks, every 3weeks, and may provide for follow-up therapy of less frequentadministration, e.g. monthly, semi-monthly, bi-monthly, etc.

In some embodiment a therapeutic regimen for treatment of cancercomprises administration of a loading dose an anti-CD47 antibody,including without limitation 5F9-G4, where the loading dose isadministered twice weekly at a dose of from 10 mg/kg to 40 mg/kg; andmay be administered twice weekly at a dose of from 20 mg/kg to 30 mg/kg.The patient is then administered a maintenance dose, weekly orsemi-weekly, at a dose of from 10 mg/kg to 40 mg/kg; and may be at adose of from 20 mg/kg to 30 mg/kg. In some such embodiments the canceris a solid tumor. In some such embodiments the cancer is a hematologiccancer, e.g. a leukemia, including without limitation acute myeloidleukemia.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1 . Red Blood Cells are Saturated with Antibody at Low Doses. Thegraph depicts receptor occupancy of CD47 on RBC in patients dosed withHu5F9-G4 at the dosage indicated. A dose of 1 mg/kg is sufficient tosaturate RBC binding sites.

FIG. 2 . WBC CD47 Receptor Occupancy Increases with Dose Concentration.The graph depicts receptor occupancy of CD47 on WBC in patients dosedwith Hu5F9-G4 at the dosage indicated.

FIG. 3A-3C. WBC CD47 Receptor Occupancy Increases with DoseConcentration. The graphs depict receptor occupancy of CD47 on WBC atvarying doses of antibody.

FIG. 4 provides graphs showing target trough levels of anti-CD47antibody for clinical use.

FIG. 5 is a graph showing pharmacokinetics of target trough levels ofanti-CD47 antibody.

FIG. 6 is a graph showing that target trough levels are approached withrepeated dosing at 10 mg/kg in human patients.

FIG. 7 is a graph showing anemia with compensatory reticulocytosisfollowing administration of anti-CD47 antibody.

FIG. 8A-8B. Hemagglutination is mitigated with a prolonged infusion timeof the priming dose. shows peripheral smear micrographs and graphs forhemagglutination associated with an initial infusion of an anti-CD47antibody. FIG. 8A. Representative peripheral smear micrographs takenfrom patients pre-treatment and 4 hours post first 1 mg/kg (priming)dose of Hu5F9-G4. Significant hemagglutination is observed in the 1 hourinfusion which is significantly reduced in the 3 hour infusion. FIG. 8B.Extension of the priming dose infusion duration from 1 to 3 hourssignificantly reduces the frequency and severity of hemagglutination. 1+to 3+ represents the percentage of agglutinated red blood cells observedon peripheral smear at 4 hours post-treatment. N=number of patientstreated with each infusion time length.

FIG. 9 . Hu5F9-G4 can achieve target PK levels at clinically feasibledoses. The data show Hu5F9-G4 can saturate the internal CD47 tissue sinkat clinically feasible doses; and antibody half-life is extended aftersaturation of the tissue sink has occurred.

DETAILED DESCRIPTION

The present invention relates to methods of treating a subject with atherapeutic dose of anti-CD47 agent.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limit of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

Anti-CD47 agent. As used herein, the term “anti-CD47 agent” refers toany agent that reduces the binding of CD47 (e.g., on a target cell) toSIRPα (e.g., on a phagocytic cell). For the specific methods of thepresent invention, agents that bind to CD47 are of interest.Non-limiting examples of suitable anti-CD47 reagents include SIRPαreagents, including without limitation high affinity SIRPα polypeptidesand anti-CD47 antibodies or antibody fragments. An agent for use in themethods of the invention will up-regulate phagocytosis by at least 10%(e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 100%, at least120%, at least 140%, at least 160%, at least 180%, or at least 200%)compared to phagocytosis in the absence of the agent. In someembodiments, the anti-CD47 agent does not activate CD47 upon binding.When CD47 is activated, a process akin to apoptosis (i.e., programmedcell death) may occur (Manna and Frazier, Cancer Research, 64,1026-1036, Feb. 1, 2004). Thus, in some embodiments, the anti-CD47 agentdoes not directly induce cell death of a CD47-expressing cell.

Anti-CD47 antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds CD47 (i.e., an anti-CD47 antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). In someembodiments, a suitable anti-CD47 antibody does not activate CD47 uponbinding. Non-limiting examples of suitable antibodies include clonesB6H12, 5F9, 8B6, and C3 (for example as described in InternationalPatent Publication WO 2011/143624, herein specifically incorporated byreference). Clone CC-9002 is disclosed in WO2013119714, hereinspecifically incorporated by reference. Suitable anti-CD47 antibodiesinclude fully human, humanized or chimeric versions of such antibodies.Humanized antibodies (e.g., hu5F9-G4) are especially useful for in vivoapplications in humans due to their low antigenicity. Similarlycaninized, felinized, etc. antibodies are especially useful forapplications in dogs, cats, and other species respectively. Antibodiesof interest include humanized antibodies, or caninized, felinized,equinized, bovinized, porcinized, etc., antibodies, and variantsthereof.

SIRPα reagent. A SIRPα reagent comprises the portion of SIRPα that issufficient to bind CD47 at a recognizable affinity, which normally liesbetween the signal sequence and the transmembrane domain, or a fragmentthereof that retains the binding activity. For the specific methods ofthe present invention, multivalent SIRPα polypeptides are of interest.

A suitable SIRPα reagent reduces (e.g., blocks, prevents, etc.) theinteraction between the native proteins SIRPα and CD47. The SIRPαreagent will usually comprise at least the d1 domain of SIRPα. In someembodiments, a SIRPα reagent is a fusion protein, e.g., fused in framewith a second polypeptide. In some embodiments, the second polypeptideis capable of increasing the size of the fusion protein, e.g., so thatthe fusion protein will not be cleared from the circulation rapidly. Insome embodiments, the second polypeptide is part or whole of animmunoglobulin Fc region. The Fc region aids in phagocytosis byproviding an “eat me” signal, which enhances the block of the “don't eatme” signal provided by the high affinity SIRPα reagent. In otherembodiments, the second polypeptide is any suitable polypeptide that issubstantially similar to Fc, e.g., providing increased size,multimerization domains, and/or additional binding or interaction withIg molecules. A SIRPα agent of interest includes TTI-621, see clinicaltrial identifier NCT02663518, herein specifically incorporated byreference.

In some embodiments, a subject anti-CD47 agent is a “high affinity SIRPαreagent”, which includes SIRPα-derived polypeptides and analogs thereof.High affinity SIRPα reagents are described in international applicationPCT/US13/21937, which is hereby specifically incorporated by reference.High affinity SIRPα reagents are variants of the native SIRPα protein.In some embodiments, a high affinity SIRPα reagent is soluble, where thepolypeptide lacks the SIRPα transmembrane domain and comprises at leastone amino acid change relative to the wild-type SIRPα sequence, andwherein the amino acid change increases the affinity of the SIRPαpolypeptide binding to CD47, for example by decreasing the off-rate byat least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold,at least 500-fold, or more.

A high affinity SIRPα reagent comprises the portion of SIRPα that issufficient to bind CD47 at a recognizable affinity, e.g., high affinity,which normally lies between the signal sequence and the transmembranedomain, or a fragment thereof that retains the binding activity. Thehigh affinity SIRPα reagent will usually comprise at least the d1 domainof SIRPα with modified amino acid residues to increase affinity. In someembodiments, a SIRPα variant of the present invention is a fusionprotein, e.g., fused in frame with a second polypeptide. In someembodiments, the second polypeptide is capable of increasing the size ofthe fusion protein, e.g., so that the fusion protein will not be clearedfrom the circulation rapidly. In some embodiments, the secondpolypeptide is part or whole of an immunoglobulin Fc region. The Fcregion aids in phagocytosis by providing an “eat me” signal, whichenhances the block of the “don't eat me” signal provided by the highaffinity SIRPα reagent. In other embodiments, the second polypeptide isany suitable polypeptide that is substantially similar to Fc, e.g.,providing increased size, multimerization domains, and/or additionalbinding or interaction with Ig molecules. The amino acid changes thatprovide for increased affinity are localized in the d1 domain, and thushigh affinity SIRPα reagents comprise a d1 domain of human SIRPα, withat least one amino acid change relative to the wild-type sequence withinthe d1 domain. Such a high affinity SIRPα reagent optionally comprisesadditional amino acid sequences, for example antibody Fc sequences;portions of the wild-type human SIRPα protein other than the d1 domain,including without limitation residues 150 to 374 of the native proteinor fragments thereof, usually fragments contiguous with the d1 domain;and the like. High affinity SIRPα reagents may be monomeric ormultimeric, i.e. dimer, trimer, tetramer, etc.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect can be prophylactic in terms ofcompletely or partially preventing a disease or symptom(s) thereofand/or may be therapeutic in terms of a partial or completestabilization or cure for a disease and/or adverse effect attributableto the disease. The term “treatment” encompasses any treatment of adisease in a mammal, particularly a human, and includes: (a) preventingthe disease and/or symptom(s) from occurring in a subject who may bepredisposed to the disease or symptom but has not yet been diagnosed ashaving it; (b) inhibiting the disease and/or symptom(s), i.e., arrestingtheir development; or (c) relieving the disease symptom(s), i.e.,causing regression of the disease and/or symptom(s). Those in need oftreatment include those already inflicted (e.g., those with cancer,those with an infection, etc.) as well as those in which prevention isdesired (e.g., those with increased susceptibility to cancer, those withan increased likelihood of infection, those suspected of having cancer,those suspected of harboring an infection, etc.)

As used herein, a “target cell” is a cell expressing CD47 on thesurface, where masking or otherwise altering the CD47 positive phenotype(e.g., by administration of an anti-CD47 agent) results in increasedphagocytosis. Usually a target cell is a mammalian cell, for example ahuman cell.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,are used interchangeably herein and refer to any mammalian subject forwhom diagnosis, treatment, or therapy is desired, particularly humans.“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc.Preferably, the mammal is human.

A “therapeutically effective dose” or “therapeutic dose” is an amountsufficient to effect desired clinical results (i.e., achieve therapeuticefficacy). A therapeutically effective dose can be administered in oneor more administrations. For purposes of this invention, atherapeutically effective dose of an anti-CD47 agent is an amount thatis sufficient to palliate, ameliorate, stabilize, reverse, prevent, slowor delay the progression of the disease state (e.g., cancer) byincreasing phagocytosis of a target cell (e.g., a target cell). Thus, atherapeutically effective dose of an anti-CD47 agent reduces the bindingof CD47 on an target cell, to SIRPα on a phagocytic cell, at aneffective dose for increasing the phagocytosis of the target cell.

In some embodiments, a therapeutically effective dose leads to sustainedserum levels of anti-CD47 agent, i.e. trough levels (e.g., an anti-CD47antibody) of about 40 μg/ml or more (e.g, about 50 μg/ml or more, about60 μg/ml or more, about 75 μg/ml or more, about 100 μg/ml or more, about125 μg/ml or more, or about 150 μg/ml or more). In some embodiments, atherapeutically effective dose leads to sustained serum levels ofanti-CD47 agent (e.g., an anti-CD47 antibody) that range from about 40μg/ml to about 300 μg/ml (e.g, from about 40 μg/ml to about 250 μg/ml,from about 40 μg/ml to about 200 μg/ml, from about 40 μg/ml to about 150g/ml, from about 40 μg/ml to about 100 μg/ml, from about 50 μg/ml toabout 300 μg/ml, from about 50 μg/ml to about 250 μg/ml, from about 50μg/ml to about 200 μg/ml, from about 50 g/ml to about 150 μg/ml, fromabout 75 μg/ml to about 300 μg/ml from about 75 μg/ml to about 250μg/ml, from about 75 μg/ml to about 200 μg/ml, from about 75 μg/ml toabout 150 μg/ml, from about 100 μg/ml to about 300 μg/ml, from about 100μg/ml to about 250 μg/ml, or from about 100 μg/ml to about 200 μg/ml).In some embodiments, a therapeutically effective dose for treating solidtumors leads to sustained serum levels of anti-CD47 agent (e.g., ananti-CD47 antibody) of about 100 μg/ml or more, e.g., sustained serumlevels that range from about 100 g/ml to about 500 μg/ml, from about 100μg/ml to about 400 μg/ml, from about 100 μg/ml to about 300 μg/ml, fromabout 100 μg/ml to about 200 μg/ml.

Accordingly, series of therapeutically effective doses would be able toachieve and maintain a serum level of anti-CD47 agent. A therapeuticallyeffective dose of an anti-CD47 agent can depend on the specific agentused, but is usually about 5 mg/kg body weight or more (e.g., about 8mg/kg or more, about 10 mg/kg or more, about 15 mg/kg or more, about 20mg/kg or more, about 25 mg/kg or more, about 30 mg/kg or more, about 35mg/kg or more, or about 40 mg/kg or more), or from about 10 mg/kg toabout 40 mg/kg (e.g., from about 10 mg/kg to about 35 mg/kg, or fromabout 10 mg/kg to about 30 mg/kg). The dose required to achieve and/ormaintain a particular serum level is proportional to the amount of timebetween doses and inversely proportional to the number of dosesadministered. Thus, as the frequency of dosing increases, the requireddose decreases. The optimization of dosing strategies will be readilyunderstood and practiced by one of ordinary skill in the art.

In some embodiments, a priming dose is defined a dose (i.e., an amount)that is sufficient to cause compensatory reticulocytosis, without undueanemia. In some embodiments a priming dose is defined as a dose thatcauses an anemia that is not worsened by subsequent doses. A primingdose of an anti-CD47 agent can depend on the specific agent used, but isgenerally from about 0.5 to about 5 mg/kg.

The term “priming dose” or as used herein refers to a dose of ananti-CD47 agent that primes a subject for administration of atherapeutically effective dose of anti-CD47 agent such that thetherapeutically effective dose does not result in a severe loss of RBCs(reduced hematocrit or reduced hemoglobin). The specific appropriatepriming dose of an anti-CD47 agent can vary depending on the nature ofthe agent used and on numerous subject-specific factors (e.g., age,weight, etc.). Examples of suitable priming doses of an anti-CD47 agentinclude from about 0.5 mg/kg to about 5 mg/kg, from about 0.5 mg/kg toabout 4 mg/kg, from about 0.5 mg/kg to about 3 mg/kg, from about 1 mg/kgto about 5 mg/kg, from about 1 mg/kg to about 4 mg/kg, from about 1mg/kg to about 3 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg,about 4 mg/kg, about 5 mg/kg.

A “maintenance dose” is a dose intended to be a therapeuticallyeffective dose. For example, in experiments to determine thetherapeutically effective dose, multiple different maintenance doses maybe administered to different subjects. As such, some of the maintenancedoses may be therapeutically effective doses and others may besub-therapeutic doses.

A “loading dose” may be used to achieve a therapeutic level of antibodybefore switching to a maintenance dose. A loading dose can be the samebe the same or higher or lower than the maintenance dose, but willgenerally provide for a higher overall delivery of the agent over agiven period of time. For example, a loading dose can be the same orlower than a maintenance dose, but delivered more frequently, e.g.daily, every other day, every third day, twice weekly, weekly, and thelike. Alternatively a loading dose can be a higher dose than amaintenance dose, and delivered at the same periodicity, or morefrequently, e.g. daily, every other day, every third day, twice weekly,weekly, and the like.

The terms “specific binding,” “specifically binds,” and the like, referto non-covalent or covalent preferential binding to a molecule relativeto other molecules or moieties in a solution or reaction mixture (e.g.,an antibody specifically binds to a particular polypeptide or epitoperelative to other available polypeptides, or binding of a SIRPαpolypeptide). In some embodiments, the affinity of one molecule foranother molecule to which it specifically binds is characterized by aK_(D) (dissociation constant) of 10⁻⁵ M or less (e.g., 10⁻⁶ M or less,10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ Mor less, 10⁻¹² M or less, 10⁻¹³ M or less, 10⁻¹⁴ M or less, 10⁻¹⁵ M orless, or 10⁻¹⁶ M or less). “Affinity” refers to the strength of binding,increased binding affinity being correlated with a lower K_(D).

The term “specific binding member” as used herein refers to a member ofa specific binding pair (i.e., two molecules, usually two differentmolecules, where one of the molecules, e.g., a first specific bindingmember, through non-covalent means specifically binds to the othermolecule, e.g., a second specific binding member). Suitable specificbinding members include agents that specifically bind CD47 and/or SIRPα(i.e., anti-CD47 agents), or that otherwise block the interactionbetween CD47 and SIRPα.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The terms “phagocytic cells” and “phagocytes” are used interchangeablyherein to refer to a cell that is capable of phagocytosis. There arethree main categories of phagocytes: macrophages, mononuclear cells(histiocytes and monocytes); polymorphonuclear leukocytes (neutrophils)and dendritic cells.

The term “sample” with respect to a patient encompasses blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived or isolatedtherefrom and the progeny thereof. The definition also includes samplesthat have been manipulated in any way after their procurement, such asby treatment with reagents; washed; or enrichment for certain cellpopulations, such as cancer cells. The definition also includes samplesthat have been enriched for particular types of molecules, e.g., nucleicacids, polypeptides, etc.

The term “biological sample” encompasses a clinical sample, and alsoincludes tissue obtained by surgical resection, tissue obtained bybiopsy, cells in culture, cell supernatants, cell lysates, tissuesamples, organs, bone marrow, blood, plasma, serum, and the like. A“biological sample” includes a sample comprising target cells or normalcontrol cells or suspected of comprising such cells or biological fluidsderived therefrom (e.g., cancerous cell, infected cell, etc.), e.g., asample comprising polynucleotides and/or polypeptides that is obtainedfrom such cells (e.g., a cell lysate or other cell extract comprisingpolynucleotides and/or polypeptides). A biological sample comprising aninflicted cell from a patient can also include non-inflicted cells.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity. “Antibodies” (Abs) and“immunoglobulins” (Igs) are glycoproteins having the same structuralcharacteristics. While antibodies exhibit binding specificity to aspecific antigen, immunoglobulins include both antibodies and otherantibody-like molecules which lack antigen specificity. Polypeptides ofthe latter kind are, for example, produced at low levels by the lymphsystem and at increased levels by myelomas.

“Antibody fragment”, and all grammatical variants thereof, as usedherein are defined as a portion of an intact antibody comprising theantigen binding site or variable region of the intact antibody, whereinthe portion is free of the constant heavy chain domains (i.e. CH2, CH3,and CH4, depending on antibody isotype) of the Fc region of the intactantibody. Examples of antibody fragments include Fab, Fab′, Fab′-SH,F(ab′)₂, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single chain polypeptide”),including without limitation (1) single-chain Fv (scFv) molecules (2)single chain polypeptides containing only one light chain variabledomain, or a fragment thereof that contains the three CDRs of the lightchain variable domain, without an associated heavy chain moiety (3)single chain polypeptides containing only one heavy chain variableregion, or a fragment thereof containing the three CDRs of the heavychain variable region, without an associated light chain moiety and (4)nanobodies comprising single Ig domains from non-human species or otherspecific single-domain binding modules; and multispecific or multivalentstructures formed from antibody fragments. In an antibody fragmentcomprising one or more heavy chains, the heavy chain(s) can contain anyconstant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fcregion of an intact antibody, and/or can contain any hinge regionsequence found in an intact antibody, and/or can contain a leucinezipper sequence fused to or situated in the hinge region sequence or theconstant domain sequence of the heavy chain(s).

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Methods

Receptor occupancy (RO) assay measures the level of CD47 occupancy byCD47 binding agents, e.g. anti-CD47 antibody (Ab). The purpose ofmeasuring the level of CD47 RO is to determine the relationship betweenthe dose of a CD47 binding agent, the CD47 receptor saturation, andpharmacologic effect. The percent of receptor occupancy over time mayprovide useful information regarding the amount of drug or duration ofexposure needed to produce the desired pharmacological effect. Thisassay can be used to determine the overall RO in the body by measuringthe CD47 RO on surrogate cells, e.g. on CD45 negative (−) red bloodcells (RBCs) and CD45 positive (+) white blood cells (WBCs), or othercell populations, e.g. bone marrow or tissue cells obtained throughtissue biopsies. The RO assay can also be used to determine CD47 RO ontarget cells, e.g. RBC, leukemia cells or solid tumor cells, for CD47binding and or blocking therapies.

Of interest is the use of this assay to determine the threshold of CD47receptor occupancy that is correlated with the desired pharmacologicaleffect. This threshold can be determined by assays performed ex vivo (invitro) or by analysis of samples during in vivo dosing/treatment.

In one embodiment of the assay, a CD47 binding standard curve on a cellof interest cells is made by using fluorochrome-conjugated antibody atvarious concentrations. Receptor occupancy is measured by incubating thetarget cells with unlabeled antibody under different concentrations, andthen the cells were either assayed in in vitro phagocytosis or incubatedwith a saturating concentration of labeled antibody based on thestandard curve and analyzed for binding by flow cytometry. Receptoroccupancy was calculated as follows:

%RO=100−((MFI_(test)−MFI_(unstained))/(MFI_(saturated STD)−MFI_(unstained)))×100

In other embodiments the assay is performed by infusing a patient with adefined dose of antibody, obtaining a tissue sample, e.g. a bloodsample, from the patient, usually before and after infusion of theantibody. The tissue sample is incubated with a saturating concentrationof labeled antibody, and analyzed by flow cytometry. The analysis may begated, for example, on red blood cells, white blood cells, cancer cells,etc.

It has been found that a priming dose that achieves at least about 80%saturation of CD47 on RBC is sufficient to induce compensation foranemia and reduce degree of anemia on subsequent doses. In humans, thepriming dose has been found to be as discussed above, i.e. from about0.5 mg/kg to about 5 mg/kg. In some embodiments of the invention, areceptor occupancy assay is performed with a candidate CD47 bind agentto determine the level of priming dose that provides for at least about50% saturation on RBC, at least about 60% saturation, at least about 70%saturation, at least about 80% saturation, at least about 90%saturation, at least about 95% saturation, at least about 99%saturation, or more.

In some embodiments of the invention, a receptor occupancy assay isperformed to determine the appropriate priming dose for a candidateanti-CD47 agent, e.g. an antibody that binds to CD47, a SIRPαpolypeptide, etc.

Treatment Methods

Methods are provided for treating a subject with a therapeutic dose ofanti-CD47 agent. The subject methods include a step of administering aprimer agent to subject, followed by a step of administering atherapeutically effective dose of an anti-CD47 agent to the subject. Insome embodiments, the step of administering a therapeutically effectivedose is performed after at least about 3 days (e.g., at least about 4days, at least about 5 days, at least about 6 days, at least about 7days, at least about 8 days, at least about 9 days, or at least about 10days) after beginning the administration of a primer agent. This periodof time is, for example, sufficient to provide for enhanced reticulocyteproduction by the individual.

The administration of a therapeutically effective dose of an anti-CD47agent can be achieved in a number of different ways. In some cases, twoor more therapeutically effective doses are administered after a primeragent is administered. Suitable administration of a therapeuticallyeffective dose can entail administration of a single dose, or can entailadministration of doses daily, semi-weekly, weekly, once every twoweeks, once a month, annually, etc. In some cases, a therapeuticallyeffective dose is administered as two or more doses of escalatingconcentration (i.e., increasing doses), where (i) all of the doses aretherapeutic doses, or where (ii) a sub-therapeutic dose (or two or moresub-therapeutic doses) is initially given and therapeutic doses areachieved by said escalation. As one non-limiting example to illustrateescalating concentration (i.e., increasing doses), a therapeuticallyeffective dose can be administered weekly, beginning with asub-therapeutic dose (e.g., a dose of 5 mg/kg), and each subsequent dosecan be increased by a particular increment (e.g., by 5 mg/kg), or byvariable increments, until a therapeutic dose (e.g., 30 mg/kg) isreached, at which point administration may cease or may continue (e.g.,continued therapeutic doses, e.g., doses of 30 mg/kg). As anothernon-limiting example to illustrate escalating concentration (i.e.,increasing doses), a therapeutically effective dose can be administeredweekly, beginning with a therapeutic dose (e.g., a dose of 10 mg/kg),and each subsequent dose can be increased by a particular increment(e.g., by 10 mg/kg), or by variable increments, until a therapeutic dose(e.g., 30 mg/kg, 100 mg/ml, etc.) is reached, at which pointadministration may cease or may continue (e.g., continued therapeuticdoses, e.g., doses of 30 mg/kg, 100 mg/ml, etc.). In some embodiments,administration of a therapeutically effective dose can be a continuousinfusion and the dose can altered (e.g., escalated) over time.

Dosage and frequency may vary depending on the half-life of theanti-CD47 agent in the patient. It will be understood by one of skill inthe art that such guidelines will be adjusted for the molecular weightof the active agent, e.g. in the use of antibody fragments, in the useof antibody conjugates, in the use of SIRPα reagents, in the use ofsoluble CD47 peptides etc. The dosage may also be varied for localizedadministration, e.g. intranasal, inhalation, etc., or for systemicadministration, e.g. i.m., i.p., i.v., s.c., and the like.

Effective administration of primer agent. An initial dose of a CD47binding agent, including but not limited to a priming dose, may lead tohemagglutination for a period of time immediately following infusion.Without being bound by the theory, it is believed that the initial doseof a multivalent CD47 binding agent may cause cross-linking of RBC boundto the agent. In certain embodiments of the invention, a CD47 bindingagent is infused to a patient in an initial dose, and optionally insubsequent doses, over a period of time and/or concentration thatreduces the possibility of hematologic microenvironments where there isa high local concentration of RBC and the agent.

In some embodiments of the invention, an initial dose of a CD47 bindingagent is infused over a period of at least about 2 hours, at least about2.5 hours, at least about 3 hours, at least about 3.5 hours, at leastabout 4 hours, at least about 4.5 hours, at least about 5 hours, atleast about 6 hours or more. In some embodiments an initial dose isinfused over a period of time from about 2.5 hours to about 6 hours; forexample from about 3 hours to about 4 hours. In some such embodiments,the dose of agent in the infusate is from about 0.05 mg/ml to about 0.5mg/ml; for example from about 0.1 mg/ml to about 0.25 mg/ml.

In other embodiments, an initial dose of a CD47 binding agent, e.g. apriming dose, is administered by continuous fusion, e.g. as an osmoticpump, delivery patch, etc., where the dose is administered over a periodof at least about 6 hours, at least about 12 hours, at least about 24hours, at least about 2 days, at least about 3 days. Many such systemsare known in the art. For example DUROS technology, provides abi-compartment system separated by a piston. One of the compartmentsconsists of osmotic engine specifically formulated with an excess ofsolid NaCl, such that it remains present throughout the delivery periodand results in a constant osmotic gradient. It also consists of a semipermeable membrane on one end through which water is drawn into theosmotic engine and establishes a large and constant osmotic gradientbetween the tissue water and the osmotic engine. Other compartmentconsists of a drug solution with an orifice from which the drug isreleased due to the osmotic gradient. This helps to provide sitespecific and systemic drug delivery when implanted in humans. Thepreferred site of implantation is subcutaneous placement in the insideof the upper arm.

Following administration of the priming agent, and allowing a period oftime effective for an increase in reticulocyte production, a therapeuticdose of an anti-CD47 agent is administered. The therapeutic dose can beadministered in number of different ways. In some embodiments, two ormore therapeutically effective doses are administered after a primeragent is administered, e.g. in a weekly dosing schedule. In someembodiments a therapeutically effective dose of an anti-CD47 agent isadministered as two or more doses of escalating concentration, in othersthe doses are equivalent. There is reduced hemagglutination after thepriming dose, and therefore the extended infusion time is not required.

Kits

Also provided are kits for use in the methods. The subject kits includea primer agent and an anti-CD47 agent. In some embodiments, a kitcomprises two or more primer agents. In some embodiments, a kitcomprises two or more anti-CD47 agents. In some embodiments, a primeragent is provided in a dosage form (e.g., a priming dosage form). Insome embodiments, a primer agent is provided in two or more differentdosage forms (e.g., two or more different priming dosage forms). In someembodiments, an anti-CD47 agent is provided in a dosage form (e.g., atherapeutically effective dosage form). In some embodiments, ananti-CD47 agent is provided in two or more different dosage forms (e.g.,two or more different therapeutically effective dosage forms). In thecontext of a kit, a primer agent and/or an anti-CD47 agent can beprovided in liquid or sold form in any convenient packaging (e.g., stickpack, dose pack, etc.).

In addition to the above components, the subject kits may furtherinclude (in certain embodiments) instructions for practicing the subjectmethods. These instructions may be present in the subject kits in avariety of forms, one or more of which may be present in the kit. Oneform in which these instructions may be present is as printedinformation on a suitable medium or substrate, e.g., a piece or piecesof paper on which the information is printed, in the packaging of thekit, in a package insert, and the like. Yet another form of theseinstructions is a computer readable medium, e.g., diskette, compact disk(CD), flash drive, and the like, on which the information has beenrecorded. Yet another form of these instructions that may be present isa website address which may be used via the internet to access theinformation at a removed site.

Utility. The subject methods and kits can be used to treat anyinfliction where the target cells exhibit an increased expression ofCD47 relative to normal cells of the same type. The anti-CD47 agent thatis administered inhibits the interaction between SIRPα (e.g., on aphagocyte) and CD47 on an target cell (e.g., on a cancer cell, on aninfected cell, etc.), thereby increasing in vivo phagocytosis of thetarget cell. Subject methods include administering to a subject in needof treatment a therapeutically effective dose of an anti-CD47 agent,including without limitation combinations of the reagent with anotherdrug (e.g., an anti-cancer drug, etc.).

The term “cancer”, as used herein, refers to a variety of conditionscaused by the abnormal, uncontrolled growth of cells. Cells capable ofcausing cancer, referred to as “cancer cells”, possess characteristicproperties such as uncontrolled proliferation, immortality, metastaticpotential, rapid growth and proliferation rate, and/or certain typicalmorphological features. A cancer can be detected in any of a number ofways, including, but not limited to, detecting the presence of a tumoror tumors (e.g., by clinical or radiological means), examining cellswithin a tumor or from another biological sample (e.g., from a tissuebiopsy), measuring blood markers indicative of cancer, and detecting agenotype indicative of a cancer. However, a negative result in one ormore of the above detection methods does not necessarily indicate theabsence of cancer, e.g., a patient who has exhibited a complete responseto a cancer treatment may still have a cancer, as evidenced by asubsequent relapse.

The term “cancer” as used herein includes carcinomas, (e.g., carcinomain situ, invasive carcinoma, metastatic carcinoma) and pre-malignantconditions, i.e. neomorphic changes independent of their histologicalorigin. The term “cancer” is not limited to any stage, grade,histomorphological feature, invasiveness, aggressiveness or malignancyof an affected tissue or cell aggregation. In particular stage 0 cancer,stage I cancer, stage II cancer, stage III cancer, stage IV cancer,grade I cancer, grade II cancer, grade III cancer, malignant cancer andprimary carcinomas are included.

Cancers and cancer cells that can be treated include, but are notlimited to, hematological cancers, including leukemia, lymphoma andmyeloma, and solid cancers, including for example tumors of the brain(glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma,ependymomas), carcinomas, e.g. carcinoma of the lung, liver, thyroid,bone, adrenal, spleen, kidney, lymph node, small intestine, pancreas,colon, stomach, breast, endometrium, prostate, testicle, ovary, skin,head and neck, and esophagus.

In an embodiment, the cancer is a hematological cancer. In anembodiment, the hematological cancer is a leukemia. In anotherembodiment, the hematological cancer is a myeloma. In an embodiment, thehematological cancer is a lymphoma.

In an embodiment, the leukemia is selected from acute myeloid leukemia(AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia(CLL) and chronic myelogenous leukemia (CML). In an embodiment, theleukemia is AML. In an embodiment, the leukemia is ALL. In anembodiment, the leukemia is CLL. In a further embodiment, the leukemiais CML. In an embodiment, the cancer cell is a leukemic cell, forexample, but not limited to, an AML cell, an ALL cell, a CLL cell or aCML cell.

Suitable cancers responsive to treatment using an anti-CD47 agentinclude without limitation leukemia; acute myeloid leukemia (AML); acutelymphoblastic leukemia (ALL); metastasis; minimal residual disease;solid tumor cancers, e.g., breast, bladder, colon, ovarian,glioblastoma, leiomyosarcoma, and head & neck squamous cell carcinomas;etc. For examples, see: (i) Willingham et al., Proc Natl Acad Sci USA.2012 Apr. 24; 109(17):6662-7: “The CD47-signal regulatory protein alpha(SIRPα) interaction is a therapeutic target for human solid tumors”;(ii) Edris et al., Proc Natl Acad Sci USA. 2012 Apr. 24;109(17):6656-61: “Antibody therapy targeting the CD47 protein iseffective in a model of aggressive metastatic leiomyosarcoma”; and (iii)US patent application 20110014119; all of which are herein incorporatedin their entirety.

Pharmaceutical Compositions. Suitable anti-CD47 agents and/or primeragents can be provided in pharmaceutical compositions suitable fortherapeutic use, e.g. for human treatment. In some embodiments,pharmaceutical compositions of the present invention include one or moretherapeutic entities of the present invention or pharmaceuticallyacceptable salts, esters or solvates thereof. In some other embodiments,the use of an anti-CD47 agent or primer agent includes use incombination with another therapeutic agent (e.g., another anti-infectionagent or another anti-cancer agent). Therapeutic formulations comprisingone or more anti-CD47 agents and/or primer agents of the invention areprepared for storage by mixing the anti-CD47 agent or primer agenthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. The anti-CD47 agent orprimer agent composition will be formulated, dosed, and administered ina fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

An anti-CD47 agent or primer agent is often administered as apharmaceutical composition comprising an active therapeutic agent andanother pharmaceutically acceptable excipient. The preferred formdepends on the intended mode of administration and therapeuticapplication. The compositions can also include, depending on theformulation desired, pharmaceutically-acceptable, non-toxic carriers ordiluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient. The pharmaceutical compositions are generally formulated assterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Toxicity of the anti-CD47 agents and/or primer agents can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., by determining the LD₅₀ (the dose lethal to 50% of thepopulation) or the LD₁₀₀ (the dose lethal to 100% of the population).The dose ratio between toxic and therapeutic effect is the therapeuticindex. The data obtained from these cell culture assays and animalstudies can be used in further optimizing a therapeutic dosage rangeand/or a priming dosage range for use in humans. The exact formulation,route of administration and dosage can be chosen by the individualphysician in view of the patient's condition.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

Example 1

Assay for Receptor Occupancy and Determination of Effective Dosage

Hu5F9-G4 serum concentrations between 50-250 μg/ml correlate withtherapeutic efficacy in AML and solid tumors in xenograft mouse models.Twice weekly maintenance doses of 10 mg/kg achieve Hu5F9-G4 serumconcentrations in the potentially therapeutic range in non-humanprimates. Weekly maintenance doses of 10 mg/kg achieve Hu5F9-G4 serumconcentrations in the potentially therapeutic range in patients.

Receptor occupancy was measured by incubating the target cells withunlabeled Hu5F9-G4 under different concentrations, and then the cellswere either assayed in in vitro phagocytosis or incubated with asaturating concentration of AF488-Hu5F9-G4 based on the standard curveand analyzed for binding by flow cytometry, gated on APC/FITC doublepopulation, and calculated MFI for each sample.

% RO=100−((MFI test−MFI unstained)/(MFI saturated STD−MFIunstained))×100  receptor occupancy:

We first assessed Hu5F9-G4 administered to cynomolgus monkeys as asingle intravenous infusion at 0, 0.1, 0.3, 1, 3, 10, and 30 mg/kg inseparate individuals. All animals were evaluated for changes in clinicalsigns, food consumption, body weights, and clinical pathologyparameters. Administration of Hu5F9-G4 was generally well tolerated, andno treatment-related effects were noted on a comprehensive list ofclinical observations, food consumption, body weights, or clinicalchemistry parameters indicative of renal, hepatic, or cardiac effects.Clinical hematology assessment indicated that Hu5F9-G4 caused adose-dependent anemia associated with reticulocytosis and spherocytosisin all animals. The nadir of the anemia occurred approximately 5-7 daysafter the infusion and generally correlated with dose. The severity ofthe anemia was variable as the two animals administered 30 mg/kgexhibited different responses. Importantly, in all animals the anemiaspontaneously resolved, returning to baseline levels after approximatelytwo weeks. In all cases, no free plasma hemoglobin was detected,indicating the absence of intravascular hemolysis. No otherabnormalities of white blood cells or platelets were observed. Thus,consistent with its known function in regulating RBC phagocytosis,Hu5F9-G4 caused a transient anemia, likely due to erythrophagocytosis,but was otherwise well-tolerated.

With single-dose administration of Hu5F9-G4, pharmacokinetic datademonstrated that only the 10 and 30 mg/kg dose levels were able totransiently achieve serum levels in the range associated with efficacyin xenograft studies. This is likely due to the large antigen sink ofCD47 expressed on circulating red and white blood cells, in addition toother non-hematopoietic tissues. Based on the previously described roleof CD47 in the normal clearance of aging red blood cells, theHu5F9-G4-related anemia observed in this study was considered related tothe pharmacological action of Hu5F9-G4 binding to CD47 expressed onRBCs. The premature loss of RBCs was compensated by an ensuingreticulocytosis, and over time, the initial anemia resolved withreplacement by younger cells.

From these considerations, we conducted a separate dose-escalation studyin NHP based on the hypothesis that initial low doses would blunt theloss of RBC and stimulate production of less-susceptible young RBC,thereby facilitating tolerance of subsequent larger doses. Two animalswere enrolled into this study and dosed at one-week intervals: one withEPO pre-treatment (3, 10, 30, 100, and 300 mg/kg), and one with nopre-treatment (1, 3, 10, 30, and 100 mg/kg). In both cases, the NHPexhibited a mild anemia with initial dosing that did not worsen withrepeated administrations. In fact, the hemoglobin only reached the upperthreshold for transfusion in humans, even without EPO pretreatment.Strikingly, the animals tolerated all doses well, including 100 and 300mg/kg, with no additional blood or metabolic abnormalities. At the endof the study, both animals were euthanized, and necropsy andhistopathology analysis revealed no abnormalities.

From this dose-escalation study, we determined the pharmacokinetics ofHu5F9-G4 in NHP. Consistent with the presence of a large antigen sink ofCD47 expressed by normal tissues, the initial low doses of Hu5F9-G4 wererapidly cleared from the serum. In contrast, the higher doses ofHu5F9-G4 produced sustained serum levels indicating saturation of theantigen sink. Remarkably, the animal dosed at 300 mg/kg had a peak levelof 5 mg/ml with a sustained level of more than 1 mg/ml for at least 2weeks. These data, suggest that a priming dose followed by a largermaintenance dose regimen should be capable of achieving the sustained50-250 μg/ml serum level that was associated with potent efficacy in thepre-clinical xenograft models.

These results led us to conduct another NHP pilot study using apriming-maintenance dosing approach to model potential clinical dosingstrategies. The goal of the priming dose would be to stimulateproduction of young RBCs that would then facilitate larger maintenancedoses capable of achieving sustained serum levels. We conducted a studyin cynomolgus monkeys in which a priming dose (PD) of either 1 or 3mg/kg was administered on Day 1, followed one week later by weeklymaintenance dosing (MD) of 30 mg/kg for six weeks. All animals wereevaluated for changes in clinical observations, food consumption, bodyweights, and clinical pathology parameters. No mortalities or changes inkey clinical chemistry parameters indicative of renal, hepatic, orcardiac effects were noted. Administration of Hu5F9-G4 was welltolerated over the entire dosing course. In both cases, the priming doseresulted in mild anemia and reticulocytosis. As hypothesized, themaintenance doses were well-tolerated with no further declines inhemoglobin throughout the treatment course. By the end of the study,hemoglobin levels returned to the normal range. Pharmacokinetic analysisindicated that exposure to Hu5F9-G4 as measured by C_(max) and the areaunder the serum concentration curve (AUC₀₋₄₃) in both animals achievedsustained serum Hu5F9-G4 levels within or above the potentialtherapeutic range for the duration of the maintenance dosing period withprolonged half-life after the final dose. These results suggest thatPD1/MD30 or PD3/MD30 dosing strategies saturate the CD47 antigen sink.Collectively, these cynomolgus monkey studies demonstrated that a lowpriming dose of Hu5F9-G4 results in a modest anemia and compensatoryreticulocytosis response that enabled subsequent higher maintenancedoses of drug to be well tolerated.

A CD47 binding standard curve on human patients following treatment witha varying priming dose of Hu5F9-G4 was made by using AF488-conjugatedHu5F9-G4, as shown in FIG. 1 . It can be seen that a priming dose of 1mg/kg or more was able to saturate greater than 80% of the CD47molecules on red blood cells with the first dose in patients, andprevents RBC agglutination with subsequent doses. Dose concentrationsare shown, whereby ⅓ and 1/10 represents a first priming dose of 1 mg/kgfollowed by 3 mg/kg maintenance doses and a first priming dose of 1mg/kg followed by 10 mg/kg maintenance doses, respectively.

Shown in FIGS. 2 and 3 is the receptor occupancy results with differentpatients, showing the effect of increasing dose, and the saturation ofreceptors on white blood cells of human patients. Each line correspondsto a different patient. Dose concentrations are shown, whereby ⅓ and1/10 represents a first priming dose of 1 mg/kg followed by 3 mg/kgmaintenance doses and a first priming dose of 1 mg/kg followed by 10mg/kg maintenance doses, respectively.

Shown in FIG. 4 , in a pre-clinical xenograft mouse model, 100-200 μg/mltrough levels are the therapeutic range, with saturation of theendogenous CD47 sink. These trough levels correlated with therapeuticanti-tumor efficacy. In a non-human primate (NHP) preclinical model,following the priming dose, target trough levels of 100 μg/ml wereachieved in the 10 mg/kg cohort. The pharmacokinetic profiles arepredictive of clinical pharmacokinetics.

With weekly administration of a dose at 10 mg/kg in a human solid tumorpatient the target trough of 100 μg/kg is achieved, illustrating thepredictive power of the preclinical NHP model, when appropriatelyconverted using FDA guidelines. In non-human primates dosing is twiceweekly at 10 mg/kg (FIG. 5 ) and in humans dosing is 10 mg/kg weekly(FIG. 6 ).

Initial anemia associated with administration of the anti-CD47 antibodyhas compensatory reticulocytosis. Shown in FIG. 7 , there is an increasein the % of reticulocytes during weekly administration of Hu5F9-G4 in ahuman patient that is mainly seen after the first (priming) dose.Hu5F9-G4 is administered weekly.

Example 2 Reduction of Hemagglutination with Extended Infusion Time

Erythrocytes express CD47 on the cell surface. However aged erythrocyteslose CD47 cell surface expression and gain expression of prophagocyticsignals. The loss or blockade of CD47 on the cell surface coupled withgain of prophagocytic signals leads to phagocytic clearance oferythrocytes. As discussed in Example 1, over a period of time fromseveral days to several weeks, administration of a priming dose ofanti-CD47 antibody can compensate for an initial transient anemia causedby administration of the anti-CD47 antibody by clearing agederythrocytes and inducing reticulocytosis, where the blood population ofred blood cells shifts to younger cells that express CD47 but do nothave prophagocytic signals.

In addition to the extended effect of anemia and compensation, there canbe an acute effect of hemagglutination immediately after administrationof an anti-CD47 antibody. Without being bound by the theory, this may beattributed to very short term high concentrations of antibody and RBClocalized at the site of administration, until normal blood flowdynamics distribute the cells and antibodies more equally. At highconcentrations the antibody may bind to different RBC, thereby causingan undesirable cross-linking effect.

To reduce acute hemagglutination it is therefore desirable to administerthe antibody in a manner that reduces the immediate concentration at theinfusion site to levels where RBC are not cross-linked. In the initialprotocols, a priming dose of antibody was administered in volumes of 250ml. for doses of 0.1 mg/kg and 0.3 mg/kg; and in a volume of 500 ml. fora dose of 1 mg/kg. over a period of one hour. As shown in FIG. 8 , thisprotocol can result in undesirable hemagglutination.

In contrast, when the same dose and concentration of antibody isadministered to a patient over a period of 3 hours, there is aremarkable improvement in the level of agglutination, shown in FIG. 8 .The extended administration can be performed for therapeutic dosing,after completion of priming, but is not required as there is a reductionin aged RBCs expressing prophagocytic signals after completion of thepriming dose.

Example 3 Clinical Trial Protocol

A novel therapeutic mAb specifically binds CD47 and blocks it frominteracting with its ligand, signal regulatory protein alpha (SIRPα), onphagocytic cells. This results in the phagocytosis and elimination ofcancer cells via prophagocytic signals, which may includephosphatidylserine, calreticulin, and others. With the exception of redblood cells, normal cells generally do not express prophagocytic signalsand are unaffected by the anti-CD47 mAb. The humanized CD47 blockingmAb, Hu5F9-G4, has been developed for clinical testing and has beenadministered safely at potentially therapeutic serum levels to non-humanprimates (NHPs). Nonclinical toxicology studies were conducted in NHPs(cynomolgus and rhesus monkeys) to support intravenous administration ofHu5F9-G4 in the clinical setting. Based on the data from the GLP 8-weektoxicology study conducted in cynomolgus monkeys, the estimated safestarting dose for human trials is 0.1 mg/kg.

The pharmacokinetics (PK) and toxicokinetics (TK) of Hu5F9-G4 have beenstudied in the cynomolgus monkey in conjunction with GLP toxicologystudy. The PK and TK collected from these studies indicate that Hu5F9-G4exhibits a varied half-life (t½), ranging from 6.35 to 320 hoursfollowing single and multiple doses. The volume of distributionapproximated monkey serum volume, as expected for a monoclonal antibody.Half-life appears to increase and clearance appears to decrease withincreasing dose and with repeated dosing, suggesting saturation oftarget-mediated clearance via the endogenous CD47 cellular sink. Therewas a notable incidence of confirmed antidrug antibodies (ADA) inmonkeys, particularly for doses at or below 10 mg/kg, which appeared tocorrelate with lower concentrations of Hu5F9-G4. Nevertheless, exposurewas maintained for doses at or above 10 mg/kg throughout the duration ofthe treatment in the repeated-dose studies.

Nonclinical studies were performed in NHPs (cynomolgus and rhesusmonkeys) to support the use of Hu5F9-G4 for intravenous administrationfor the duration of 8 weeks in the clinical trial. The GLP toxicologystudy was conducted in cynomolgus monkeys, with the dosing phase being 8weeks in duration followed by an 8 week recovery period. In the GLP8-week toxicology study, Hu5F9-G4 was administered to male and femalecynomolgus monkeys via a 1-hour intravenous infusion using apriming/maintenance dose schedule for a total of 8 weeks. Hu5F9-G4 wasadministered as a priming dose (5 mg/kg) on Day 1, followed bymaintenance doses of 5, 10, 50, or 100 mg/kg administered twice weeklyon Days 8, 11, 15, 18, 22, 25, 29, 32, 36, 39, 43, 46, 50, and 53.

The primary treatment-related finding noted in this study (as well asthe previous pilot toxicology studies) was decreases in red cell mass,including red blood cell (RBC) count, hemoglobin, and hematocrit. Thedecreased hemoglobin was noted in all Hu5F9-G4-treated animals followingthe administration of the priming dose on Day 1, and the decrease inhemoglobin was generally most pronounced following administration of thefirst maintenance dose on Day 8. The severity and incidence of theHu5F9-G4-related anemia varied across animals, and while the decreasedhemoglobin did not occur in a clear dose dependent manner, the high-dosemaintenance group (100 mg/kg) had the highest incidence of animalshaving a hemoglobin level ≤10.0 g/dL on Day 11 (90%). Importantly, thehemoglobin levels showed a trend toward recovery across all animals(generally starting around Days 15-32) and continued to recover to theend of the study. Due to severe anemia (hemoglobin ≤7.0 g/dL), twoanimals (one in the 5/50 mg/kg group and one in the 5/100 mg/kg group)were placed on dose holiday to evaluate the recovery of the anemia andhow the animals responded once dosing resumed. The decrease inhemoglobin level for the animal in the 50 mg/kg maintenance dose groupwas as low as 5.7 g/dL on Days 15 and 18, and thus, had dose holidays onDay 25-36 (maintenance doses 6-9). The hemoglobin for this animal beganto recover on Day 36, and thus, dosing resumed for this animal on Day 39(Dose 10). Even though the maintenance dosing resumed, the hemoglobinlevel for this animal continued to recover until the end of the study.The hemoglobin level for the animal in the 100 mg/kg maintenance dosegroup dropped to 6.9 g/dL on Day 18, and thus was placed on dose holidayon Day 25; this animal continued on dose holiday until the end of thestudy. The hemoglobin levels for this animal also showed a continuedtrend to recovery until the end of the study.

Thus, while it appears that a small number of animals may be especiallysensitive to the anemia produced by Hu5F9-G4, no clinical signs oftoxicity were observed in these animals, and furthermore, the anemia istransient and the hemoglobin levels recover over time. In addition tothe decreases in red cell mass, increases in reticulocytes were observedin all Hu5F9-G4-treated groups, which is indicative of a robusterythropoietic response associated with the decreases in RBC mass.

Consistent with the previous studies, the decreased red cell mass wasalso associated with decreases in mean corpuscular volume (MCV) andhaptoglobin and increases in mean corpuscular hemoglobin concentration(MCHC), reticulocytes, and red cell distribution width (RDW). Notably,free plasma hemoglobin was not observed in any Hu5F9-G4-treated groups,which is indicative of an absence of intravascular hemolysis. Minimal tomild increases in lymphocytes were also observed, but these increaseswere transient and sporadic in nature and did not occur in adose-dependent manner. Changes in blood cell morphology were consideredto be associated with accelerated red blood cell destruction/clearanceand increased erythropoiesis, and included anisocytosis, spherocytes(microcytes), eccentrocytes, atypical erythrocyte fragments consistentwith erythrocyte injury/clearance, erythrocyte clumping and largeplatelets, as well as changes associated with increased erythropoiesisconsisting of anisocytosis and polychromatophilic macrocytes.

All of treatment-related changes in hematology parameters, includingblood cell morphology, showed a continued trend toward recovery to theend of the study. Changes in bone marrow smear evaluations were limitedto minimal to mild morphologic changes in the erythroid lineage(dysplasia), which consisted of occasional cells with abnormal nuclearshapes, multiple nuclei, nuclear blebbing, and/or nuclear to cytoplasmmaturation asynchrony (abnormal nucleus to cytoplasm maturation). Markedreticulocytosis was associated with the treatment-related decreases inred cell mass, which is indicative of accelerated erythropoiesis.Additional changes considered to be related to the acceleratederythropoietic response associated with Hu5F9-G4 included mild decreasesin the mean M:E ratio along with an appropriate minimal to mild shift tomore immature erythroid precursors associated with acceleratederythropoiesis. Consistent with previous studies, treatment-relatedchanges in hematology parameters (i.e., decrease hemoglobin; increasedreticulocytes) observed in the GLP 8-week study were associated withincreases in total bilirubin and decreases in haptoglobin.

Other changes in clinical chemistry parameters were observed only in the100 mg/kg maintenance dose group, and included a slight decrease inalbumin (two female animals), a slight increase in globulin, and acorresponding decrease in albumin:globulin ratio. All treatment-relatedchanges in clinical chemistry parameters were partially or completelyreversible at the end of the dosing phase.

Based on the known role of CD47 in the normal clearance of aging redblood cells, the Hu5F9-G4-related anemia observed in this study isconsidered related to the pharmacological action of Hu5F9-G4 binding toCD47 expressed on RBCs. We hypothesize that the administration ofHu5F9-G4 accelerates the process of elimination of aging RBCs bysubstituting gradual loss of CD47 with immediate blockade of CD47 onaging RBCs. The premature loss of aging RBCs is compensated by anensuing reticulocytosis (which was observed across all studies), andover time, the initial anemia resolves as the aged RBCs are replacedwith younger cells, and as a result, the age distribution of the RBCpool is shifted to younger cells.

Overall, administration of Hu5F9-G4 via a 1-hour IV infusion at apriming dose of 5 mg/kg in Week 1 (Day 1), followed by twice weeklymaintenance doses for 7 consecutive weeks at doses up to 100 mg/kg, wasclinically well tolerated in cynomolgus monkeys. Despite thetreatment-related anemia, no signs of clinical toxicity were observed,including in the animals having dose holidays due to the severe anemiaobserved. The hematology changes observed in this study were consistentwith previous studies and considered to be related to thepharmacological action of Hu5F9-G4 in accelerating the process of agingRBC elimination through binding of Hu5F9-G4 to CD47 expressed on RBCs.All treatment related changes in hematology and clinical chemistryparameters were partially or completely reversible by the end of thestudy. Therefore, based on the totality of the data, the highestnon-severely-toxic-dose (HNSTD) for this study was considered to be thepriming/maintenance dose of 5/100 mg/kg, the highest dose evaluated. Thepredicted safety margin (based on AUC) provided by the 100 mg/kgmaintenance dose is more than adequate and ranges from 766- to 803-foldabove the starting dose of 0.1 mg/kg for the proposed clinical study.

The humanized anti-CD47 antibody, Hu5F9-G4, was tested for its effectson pre-transfusion blood typing and cross-matching in preparation ofstrategies for management of patients who might need transfusion duringHu5F9-G4 treatment. Results showed that Hu5F9-G4 did not interfere withplasma antibody screening, making it possible to detect alloantibodiesand to proceed with packed red blood cell (PRBC) transfusion ifmedically required. However, in whole blood Hu5F9-G4 interfered with theresults for ABO blood typing, direct antiglobulin test (DAT), and redblood cell (RBC) immunophenotyping. Therefore, it will be important tohave ABO blood typing, DAT, and RBC immunophenotyping performed on bloodsamples obtained prior to initiation of Hu5F9-G4 treatment. Hu5F9-G4 wasincubated with RBCs from non-human primates and human donors. Hu5F9-G4did not induce in vitro hemolysis of human RBCs, even in the presence ofcomplement-containing serum. The study showed no evidence ofhemagglutination (HA) in seven NHP specimens. However, HA was observedin all 14 human donor blood samples in the presence of 10 micrograms/mLof Hu5F9-G4.

Unlike most cases of agglutination-related autoimmune hemolytic anemiawhere HA is caused by cold IgM agglutinins, the agglutination caused byHu5F9-G4, an IgG4, occurred at 37° C. but not at 4° C. While RBCagglutination can be seen in several conditions (usually in associationwith infectious agents, both with and without clinical sequelae), theclinical significance of the agglutination seen here is uncertain.

This is an open label, non-randomized, Phase 1, first-in-human,escalating dose cohort study of the CD47 blocking antibody Hu5F9-G4. Theexpected DLT of this drug is anemia, due to phagocytosis of senescenterythrocytes by macrophages. Pilot toxicology studies conducted incynomolgus monkey studies showed that administration of a priming lowdose of antibody produced a modest anemia and a reticulocyte responsethat enabled subsequent higher maintenance doses of drug to be welltolerated. Therefore, the strategy in this Phase 1 trial is to firstestablish an optimal priming dose in an escalating dose cohort design(Part A) at which 6 of 6 patients maintain a hemoglobin of 8 g/dL orgreater in the absence of transfusion for the first four weeks and thatproduces a DLT, other than anemia, in no more than 1 of 6 patients. Oncethis optimal priming dose is established in Part A, it will beadministered Day 1 of Week 1, to be followed one week later by weeklymaintenance dosing in Part B, and if necessary, by Part C (twice weeklyloading dosing for Weeks 2-4, followed by weekly maintenance dosing).The initial dose of 0.1 mg/kg in the first cohort is supported by asafety margin of 766-fold to 803-fold relative to the 100 mg/kgtwice-weekly maintenance dose, which is the HNSTD in the pivotal GLPtoxicology study. Based upon these studies, and assuming linear scalingof the safety margins (which may not be applicable), we expect thatHu5F9-G4 will be clinically tolerated at doses up to 10 mg/kg andperhaps higher. Therefore, a dose escalation schema of 0.1, 0.3, 1, 3,10, and 20 mg/kg was selected.

For Part A, for the first patient in each cohort, evaluation for DLTswill be completed on Day 29 prior to enrollment of the second patient onthe cohort. The second patient in a cohort of 3 will not be able tobegin treatment until 2 weeks after the first patient has begun the DLTassessment period (14 days from start of therapy). The third patient canbegin 4 weeks after the first patient has begun the DLT assessmentperiod (28 days from the start of therapy). For Parts B and C, for thefirst patient in each cohort, evaluation for DLTs will be completed onDay 29 prior to enrollment of the second patient on the cohort.Subsequent patients on each cohort can begin treatment two weeks afterthe preceding patient starts therapy. In addition, the third patient inthe cohort will require observation for 28 days prior to eitherproceeding to the next cohort or expansion to a cohort of 6. If a cohortis expanded from 3 to 6 patients, those patients can be treated 28 daysafter the third patient has initiated therapy, without an additionalobservation period between the fourth and sixth patients in that cohort.Dose level allocation will be decided by the CTMC.

This is a single center, open-label trial with three parts. Thefollowing apply to all parts of the study: Dose escalation will proceedthrough the designated dose levels, and decisions regarding doseescalation will be based upon the first 4 weeks of treatment in thecurrent cohort, referred to as the “Dose Limiting Toxicity (DLT)Assessment Period,” in conjunction with ongoing assessments for patientson prior cohorts who continued therapy beyond 4 weeks. Decisionsregarding additional cohorts to further refine the MTD or RP2DS will bemade by the CTMC and will require a study amendment.

Definition of Dose Limiting Toxicity (DLT). A DLT is defined as apossibly, probably, or definitely drug related adverse event (AE),occurring within the first four weeks of therapy, as follows: AE ofGrade 3 or greater with exceptions listed below; Anemia as a consequenceof the IMP is considered a Grade 3 toxicity if transfusion is indicated,irrespective of level of hemoglobin. Any transfusions or Grade 3 orgreater severity of anemia or the need to utilize erythropoiesisstimulating agents will be considered a DLT and will lead to removalfrom the study protocol. The following will not be considered DLT andare excluded from the DLT definition: Grade 3 nausea in patients whohave not received optimal treatments with antiemetics, and that resolvesto <Grade 2 within 48 hours; Grade 3 vomiting in patients who have notreceived optimal treatment with anti-emetics, and that resolves to<Grade 2 within 48 hours; Grade 3 diarrhea in patients who have notreceived optimal treatment with anti-diarrheals, and that resolves to<Grade 2 within 48 hours; Grade 3 fatigue that resolves within two weekson study; Grade 3 infusion reactions in the absence of pretreatment.Grade 3 increase in indirect/unconjugated blood bilirubin that resolvesto baseline or Grade 1 within 7 days or prior to the next scheduledHu5F9-G4 dose, whichever is sooner and the increase inindirect/unconjugated bilirubin is not temporally associated with aGrade 2 or higher increase in AST, ALT, and/or alkaline phosphatase fromhepatic source (fractionation of alkaline phosphatase to determineorigin is at the discretion of the investigator). Criteria for severitygrading of indirect/unconjugated and direct/conjugated bilirubin willuse CTCAE 4.03 criteria as it would apply to blood bilirubin. Anincrease in total bilirubin will not be a DLT if direct/conjugatedbilirubin is Grade 2 or lower and indirect/unconjugated bilirubinelevation has been determined to not be a DLT.

Definition of Maximum Tolerated Dose (MTD). The optimal priming dose forWeek 1, Day 1 in Parts B and C will be selected as the maximum dose inPart A at which 6 of 6 patients did not require blood producttransfusion and maintained a hemoglobin greater than or equal to 8 g/dL(Grade 0-2 anemia) and with no more than 1 of 6 patients with a DLTother than anemia during the first 4 weeks of treatment. The MTD forParts B and C is defined as the maximum dose level at which no more than1 of 6 patients experienced a DLT and below the dose level at which 2 ormore patients experienced a DLT for those patients who receive at leastone maintenance dose of Hu5F9-G4. AEs that occur for patients in Part Band C who do not receive at least one maintenance dose (for Part B) orat least one loading dose (for Part C) will not be included in the MTDassessment for selection of the maintenance (Part B) or loading (Part C)doses. It is possible that an MTD will not be achieved in Parts B or C,in which case the maximum administered tolerated dose will bedetermined. The maximum planned weekly maintenance dose is 20 mg/kg.

Patient Evaluability. All patients exposed to the IMP, Hu5F9-G4, will beevaluable for safety and contribute data to dose escalation decisions.Patients who (1) decline participation and do not experience a DLT, or(2) are removed from the study for reasons not related to Hu5F9-G4related AEs, or (3) have an AE not related to Hu5F9-G4 that requiresremoval from study before completion of the first four weeks of therapy,may be replaced by adding another patient to that cohort.

Drug Administration. Hu5F9-G4 will be administered intravenously. ForPart A: The durations of the IV infusions of Hu5F9-G4 will be 60 minutes(±10 minutes) for the doses from 0.1 to 1 mg/kg and two hours (±10minutes) for doses greater than 1 mg/kg.

For Parts B and C: The priming dose is the first dose that subjects inParts B and C will receive and was determined to be 1 mg/kg at thecompletion of Part A. For subjects in Parts B and C, the duration of theinfusion of the priming dose, namely 1 mg/kg, will be three hours. Themaintenance doses will be administered after the completion of thepriming dose. The duration of the infusion of the maintenance doses willbe two hours for doses greater than 1 mg/kg. Patients with dose delay ordrug holiday of 2 weeks or longer will be re-primed in which case thepriming dose of 1 mg/kg will be administered over three hours prior toresumption of the assigned maintenance dose. Premedication may beadministered prior to the second or subsequent doses for patients whoexperience an infusion reaction to previous Hu5F9-G4 administration. Thesuggested premedication regimen could include a combination ofacetaminophen 500 mg PO, dexamethasone 8 mg IV, and diphenhydramine 25mg IV administered 30 minutes prior to the infusion of Hu5F9-G4. In thecase of an infusion reaction, a premedication regimen for subsequenttreatments is at the discretion of the investigator.

Identification of Priming Dose

The overall aim of this portion of the study is to identify the primingdose that results in an acceptable level of anemia (less than Grade 3)within the first 4 weeks in all 6 patients of the dose-defining cohort.Thus, the optimal priming dose level will be the maximum dose level atwhich all 6 patients maintain a hemoglobin of 8 g/dL or greater in theabsence of transfusion for the first four weeks and which produces a DLTother than anemia in no more than 1 of 6 patients. Patients will beassessed in successive dose cohorts as follows: 0.1, 0.3, 1, 3, 10, and20 mg/kg administered as an IV infusion. Dose escalation in Part A willfollow a modified accelerated titration design for dose levels below 3mg/kg and the standard 3 plus 3 dose escalation design for dose levelsat 3 mg/kg and above. For the accelerated titration design, one patientwill be enrolled per cohort until a Grade 2 or greater AE related toHu5F9-G4 is observed within the first four weeks. Such an event willresult in cohort expansion to 3 patients. However, any occurrence ofGrade 3 anemia in the first four weeks for any patient in Part A willresult in cessation of further accrual into that cohort and will resultin expansion of the next lower dose cohort. For AEs other than anemia,one patient with DLT (Grade 3 or greater AE related to Hu5F9-G4) in thefirst three patients will result in a cohort expansion to 6 patients.Two patients with DLT indicate that the maximum tolerated priming dosehas been exceeded, further enrollment to that cohort will not bepermitted, and the next lower dose cohort, if it contains 3 or fewerpatients, will be expanded to 6 patients. Patients enrolled in Part Awill continue weekly treatment at the assigned dose level untilunacceptable toxicity or documentation of progressive disease (asdetermined by RECIST v 1.1 for solid tumors or the IWG criteria forlymphomas) or voluntary patient withdrawal from the study. Except forthe priming dose on Day 1 of Part B and C, followed by highermaintenance and/or loading doses, there will be no intra-patient doseescalation in this study. Please note that any Grade 3 anemia in thefirst 4 weeks of Part A will trigger cessation of the cohort andexpansion of the next lower dose cohort. Moreover, the acceleratedtitration design only applies to dose levels below 3 mg/kg. Dose levelsof 3 mg/kg or greater will be performed with a standard 3 plus 3 design.After completion of Part A and per protocol Amendment 4.0, the primingdose was identified as 1 mg/kg. In addition, ongoing weekly treatmentwith 1 mg/kg in Part A established that 1 mg/kg is also safe as amaintenance dose.

Expanded Safety Assessment of the Priming Dose During Parts B and C.Patients in Parts B and C will receive the optimal priming dose of 1mg/kg on Week 1, Day 1 that was determined from Part A, followed byweekly maintenance doses starting on Day 8. If an AE or SAE occurs aftera patient receives the priming dose but before the patient receives amaintenance dose then this AE or SAE will be attributed to the primingdose and contribute to the expanded safety assessment of the primingdose. SAEs and AEs attributed to the priming dose will continue to beclosely monitored by the CTMC with meetings to convene on a regularbasis or more frequently if necessary (as per the CTMC charter).

Part B: Identification of the MTD for Weekly Maintenance Dosing After aSingle Priming Dose. All patients in Part B will receive the optimalpriming dose on Week 1, Day 1 that was determined from Part A, followedby weekly maintenance doses starting on Day 8. The weekly maintenancedose administered is determined by the assigned Part B dose cohort. Theoriginal proposed dose cohorts for Part B were 0.3, 1, 3, 10, and 20mg/kg. Weekly maintenance dose cohorts will begin at the dose that isone dose level higher than the optimal priming dose. The Part A 1 mg/kgdose level was determined to be the optimal priming dose and weeklymaintenance dosing at 1 mg/kg was determined to be safe; therefore, thefirst maintenance dose level assigned in Part B will be the 3 mg/kg IVweekly dose level and maintenance dose levels 0.3 and 1 mg/kg will beskipped. Dose escalation in Part B will follow a standard 3 plus 3 doseescalation design. The MTD for Part B is the maximum dose level at whichno more than 1 of 6 patients experiences a DLT and below the dose levelat which 2 or more patients experience a DLT for those patients whoreceive at least one maintenance dose of Hu5F9-G4. AEs that occur forpatients in Part B who do not receive at least one maintenance dose ofHu5F9-G4 will not be included in the DLT assessment for selection of themaintenance dose. To further refine the MTD, a dose level midway betweenthe nominal MTD and the next higher dose level may be tested. Forexample, if two or more subjects at the 3 mg/kg dose level experience aDLT attributed to the maintenance dose, a dose level midway between 1mg/kg and 3 mg/kg, namely 2 mg/kg, may be tested to further refine theMTD allowing the final MTD determination to be increased from 1 mg/kg to2 mg/kg if no more than 1 of 6 subjects at 2 mg/kg experience a DLT at 2mg/kg. Similarly, dose levels of 6.5 mg/kg and 15 mg/kg may be added ifthe nominal MTD is 3 mg/kg or 10 mg/kg, respectively. It is possiblethat an MTD will not be achieved in Part B for the maintenance dose, inwhich case the maximum administered tolerated dose will be determinedbecause the maximum weekly dose will be 20 mg/kg. The DLT assessmentperiod for the maintenance dose on Part B will start from the time ofadministration of the first maintenance dose (Week 2, Day 8) until oneweek after the completion of the third maintenance dose (Week 4, Day29). Patients enrolled in Part B will continue weekly treatment at theassigned dose level until unacceptable toxicity or documentation ofprogressive disease as determined by RECIST v 1.1 for solid tumors, orIWG criteria for lymphomas, or voluntary patient withdrawal from thestudy. Except for the priming dose on Day 1 of Part B and C, followed byhigher maintenance and/or loading doses, there will be no intra-patientdose escalation in this study. The PK targets for the RP2DS are theachievement and maintenance of a trough level of the Hu5F9-G4 antibodyabove 100 micrograms/mL in plasma in five of six patients at the RP2DS.When the MTD or maximum administered tolerated dose (in the absence ofan MTD) or optimal biological (based on PK) dose for weeklyadministration has been determined, the study may be amended to addadditional cohorts to Part B where weekly dosing is followed by Q14and/or Q21 day dosing. The more prolonged schedules would be initiatedonly after detailed review of available safety, PK, and pharmacodynamicsdata. For example, this could be initiated when PK parameters indicatesaturation of target-mediated clearance and prolongation of Hu5F9-G4half-life. Dosing during the Q14 or Q21 day interval cohorts may beincreased to 30 mg/kg, by protocol amendment if 20 mg/kg is welltolerated.

Part C: Identification of the MTD for Twice Weekly Loading Dosing, Afteran Initial Priming Dose and Prior to Weekly Maintenance Dosing, ifAdequate PK Parameters are not Achieved in Part B by Weekly Dosing. PartC may be initiated if adequate exposure to Hu5F9-G4 is not achieved inPart B. Adequate exposure is defined as a trough level of antibody above100 micrograms/mL achieved in five of six patients at the recommendedPhase 2 dose by Day 57. All patients in Part C will receive the optimalpriming dose determined in Part A on Week 1, Day 1, followed by twiceweekly loading doses according to the assigned Part C dose cohort. SAEsand AEs attributed to the priming dose will continue to be closelymonitored by the CTMC with meetings to convene on a regular basis ormore frequently if necessary as needed (as per the CTMC charter). Theoriginal proposed dose cohorts for Part C were 0.3, 1, 3, 10, and 20mg/kg. Twice weekly loading dose cohorts will begin at the dose levelthat is one level lower than the Part B MTD or the maximum administeredtolerated dose. Thus, if 20 mg/kg was the MTD in Part B, the twiceweekly loading dose in Part C will be 10 mg/kg. Twice weekly loadingdoses (administered the first and fourth day of each weekly period) willbe administered during Weeks 2, 3, and 4. This will be followed byweekly maintenance doses in Weeks 5-8. After the completion and analysisof Hu5F9-G4 serum concentrations within the proposed cohorts in Part Cand in the event that adequate exposure is not achieved during twiceweekly administration at the end of three weeks of loading (Weeks 2-4),the protocol may be amended to include cohorts of twice weekly loadingdosing for additional 2-week periods beyond Week 4. Dose levels higherthan 20 mg/kg for either loading or maintenance in Part C will require aprotocol amendment. Similarly to Part B, additional cohorts may be addedto Part C when the MTD or maximum administered tolerated dose (in theabsence of MTD) or optimal biological dose has been determined, wheretwice weekly loading dosing, then weekly maintenance dosing, is followedby Q14 or Q21 day dosing based on safety, PK, and pharmacodynamics data.For example, a more prolonged schedule would begin at a time point whenPK parameters indicate saturation of target-mediated clearance andprolongation of Hu5F9-G4 half-life. The dose employed for the Q14 day orQ21 day cohorts may be increased to 30 mg/kg, by protocol amendment, if20 mg/kg is well tolerated. Dose escalation in Part C will follow astandard 3 plus 3 design for all dose levels. DLT assessment towards theMTD will apply for those patients who receive at least one loading dosefor Part C. DLTs that occur for patients in Part C who do not receive atleast one loading dose of Hu5F9-G4 will not be included in the DLTassessment for identification of the MTD for the loading dose. If nopatients experience a DLT, then dose escalation may proceed to the nexthigher dose level. If one of three patients experiences a DLT, then thatsame dose level cohort will be expanded to 6 patients. If 2 or more of 6patients experiences a DLT, then no further enrollment will be permittedat that dose level and the MTD will have been exceeded. The MTD for PartC is the maximum dose level at which no more than 1 of 6 patientsexperiences a DLT and below the dose level at which 2 or more of 6patients experiences a DLT. The DLT assessment period for the loadingdose on Part C will start from the time of administration of the firstloading dose (Week 2, Day 8) until Week 4, Day 29. Patients enrolled inPart C will continue treatment at the assigned dose level untilunacceptable toxicity or documentation of progressive disease asdetermined by RECIST v 1.1 for solid tumors or by IWG criteria forlymphomas or voluntary patient withdrawal from the study.

Intra-Patient Dose Escalation. Except for the priming dose on Day 1 ofPart B and C, followed by higher maintenance and/or loading doses, therewill be no intra-patient dose escalation in this study.

Investigational Agent

The active pharmaceutical ingredient (API) is Hu5F9-G4, a humanizedmonoclonal antibody of the IgG4 kappa isotype containing a Ser-Pro (S-P)substitution in the hinge region of the heavy chain to reduce Fab armexchange. It is comprised of a disulfide-linked glycosylated tetramerconsisting of two identical 444 amino acid heavy gamma chains and twoidentical 219 amino acid kappa light chains. Hu5F9-G4 targets CD47.Hu5F9-G4 drug product is provided in a liquid dosage form intended forIV infusion. Hu5F9-G4 is supplied in single-use, 10 mL vials containing200 mg of the antibody, in a formulation of 10 mM sodium Acetate, 5%(w/v) sorbitol, 0.01% (w/v) polysorbate 20, pH 5.0.

Hu5F9-G4 drug substance has been manufactured at Lonza Group, Ltd(Slough, UK), and drug product has been manufactured by Patheon UKLimited (Swindon, UK). The drug product, referred to as IMP throughoutthis document, will be supplied by the Sponsor of this trial, StanfordUniversity, via a storage and distribution contract with FisherBioServices.

Details of Administration of Hu5F9-G5

Dose Calculation. The individual dose is calculated using the actualbody weight of the patient at enrollment (using weight obtained ateither Screen or Day 1), and the dose may remain constant throughout thestudy unless a greater than 10% change in weight is observed. Thefollowing formula should be used to calculate the volume of Hu5F9-G4from the vials containing 20 mg/mL (200 mg total of Hu5F9-G4 per vial)required for each administration: Body Weight (kg)×Desired Dose(mg/kg)=Volume of Hu5F9-G4 (mL) 20 mg/mL

For Part A: For dose-escalation patients requiring a dose of 1 mg/kg orless, Hu5F9-G4 will be administered as a continuous IV infusion in 250mL over 60 minutes (±10 minutes). All other infusions for doses greaterthan 1 mg/kg will be administered in 500 mL over 2 hours (±10 minutes).

For Parts B and C: The priming dose is the first dose that patients inParts B and C will receive and was determined to be 1 mg/kg at thecompletion of Part A. SAEs and AEs attributed to the priming dose willcontinue to be closely monitored by the CTMC with meetings to convene ona regular basis or more frequently as needed (as per the CTMC charter).For subjects in Parts B and C, the duration of the infusion of thepriming dose, namely 1 mg/kg, will be three hours. In Part B, themaintenance doses will be administered starting on Day 8, one week afterthe completion of the priming dose. The duration of the infusion of themaintenance doses will be two hours for doses greater than 1 mg/kg. InPart C, the loading doses will be administered starting on Day 8, oneweek after the completion of the priming dose, and the maintenance doseswill be administered starting on Day 29. The duration of the infusion ofthe loading and maintenance doses will be two hours for doses greaterthan 1 mg/kg.

Patients in Parts B and C with dose delay or drug holiday of 2 weeks orlonger will be reprimed in which case the priming dose of 1 mg/kg willbe administered over three hours prior to resumption of the assignedmaintenance dose; for patients in Part A the re-priming infusionduration will be one hour as per protocol. Hu5F9-G4 should NOT beadministered as a bolus injection.

Adjustments to the dosing schedule will be allowed for treatment-relatedtoxicity as follows: No treatment delays are acceptable for the firstfour weeks of treatment. Thereafter, treatment may be delayed for up tothree weeks to allow sufficient time for recovery from non-DLTtreatment-related toxicities. Patients who develop Grade 2 anemia mayhave a treatment delay of up to three weeks beginning at Week 5 to allowfor recovery of the anemia to Grade 1. However, any DLT will requireremoval of the patient from the study. Treatment delays for more thanthree weeks (such as for an unrelated medical condition with expectedrecovery) must be approved by the CTMC. Additionally, drug holidays forup to 2 weeks will be allowed after Day 57 at the discretion of theinvestigator and with written Sponsor approval. “Drug Holiday” isdefined as a holiday from protocol specified treatment, assessments, andprocedures. There must be a minimum of 6 days between Hu5F9-G4 infusionsin Parts A and B and a minimum of 3 days between Hu5F9-G4 infusionsdelivered by twice weekly infusions. Patients in Parts B and C with atreatment delay or holiday of 2 weeks or more must be “reprimed” byreceiving the priming dose of 1 mg/kg IV over three hours again prior toresuming the assigned maintenance treatment dose; for patients in Part Athe re-priming infusion duration will be one hour as per protocol.

Receptor Occupancy Assay

Receptor occupancy samples will be drawn per the schedule ofassessments. Blood cells will be analyzed by flow cytometry for CD47receptor occupancy in the white blood cell and red blood cell fractions.The baseline (Day 1) sample from each patient, collected prior to thefirst Hu5F9-G4 antibody incubation, will be incubated with increasingconcentrations of Hu5F9-G4 antibody to establish a standard curve forCD47 molecule/receptor occupancy. In addition, phosphatidylserineexpression on red blood cells may be assessed by annexin V expression atbaseline (Day 1) and on Day 8, which can be completed as an additionalaspect of the receptor occupancy assay without additional blood draws.

CD47 receptor occupancy will be determined on primary cancer cells byflow cytometry or immunofluorescence on samples obtained from malignanteffusions or tissue biopsies, when available.

Changes in the immune cell compartment composition in the tumors will bedetermined in tumor biopsies collected before and after treatment, whenavailable.

Association of somatic cancer mutations with response to Hu5F9-G4. Forexample, colorectal, lung, and head and neck tumors may be analyzed forKRAS, BRAF, NRAS, and/or PIK3CA mutations.

Response to Hu5F9-G4 treatment for individual study patients utilizingin vitro assays and xenotransplantation mouse studies.

Assessment of potential resistance to Hu5F9-G4 and exploration ofalternative CD47 blocking strategies to overcome resistance (e.g., highaffinity SIRP-alpha Fc fusion proteins).

Peripheral Blood Smear Assessment: Peripheral Smears will be assessedfor the presence of hemagglutination in addition to standard cellmorphology assessment. These labs should be drawn in the armcontralateral to the drug infusion if possible. For the first 2 weeks oftreatment in Parts A and B, peripheral smears will be performed on Days1 and 8 pre-dose and 4 hours after the end of each IMP infusion (±30minutes), Days 2 and 9 at 24-hour post IMP infusion (±4 hours), Days 4and 11 at the 72-hour time point (±24 hours). In Part A after Week 2,peripheral smears will be performed pre-dose on Day 15 and thereafter atthe discretion of the investigator. In Part B after Week 2, peripheralsmears will also be performed pre-dose on Days 15 and 22, but for D15peripheral smear will also be performed 4 hours after the end of theinfusion (±30 minutes). For Days 29 and following, peripheral smearswill be performed at the discretion of the investigator and at the Endof Study Visit. In Part C, peripheral smears will be performed on Days 1and 8 pre-dose and 4 hours after the end of the IMP infusion (±30minutes), Days 2 and 9 at 24-hour post IMP infusion (±4 hours), Day 4 atthe 72-hour time point (±24 hours), and will also be performed pre-doseand at 4 hours after the end of the IMP infusion (±30 minutes) on Days11, 15, 18, 22, 25 and 29. For Days 30 and following, peripheral smearswill be performed at the discretion of the investigator and at the Endof Study Visit. For patients undergoing blood transfusion, peripheralsmears will be performed prior to blood transfusion and again 4 hours(±30 minutes) after completion of the transfusion.

Pharmacokinetic Analyses

The Safety Analysis Set will be included in the populations for PK, CD47receptor occupancy, immunogenicity, and exploratory biomarkers if dataare available for analysis. In addition, the PK population requiressufficient measureable concentration data for the estimation of PKparameters, while the PK concentration population will include allpatients with any measurable concentrations of Hu5F9-G4. The inclusionof patients with protocol violations will be assessed on apatient-by-patient basis for inclusion in the PK population prior to theanalysis. Concentration versus time data will be summarizeddescriptively, including N, Mean, SD, Geometric Mean, Median, Min, Maxand % CV. Individual and mean Hu5F9-G4 concentration versus time curveswill be graphically presented. Pharmacokinetic parameters to becalculated using non-compartmental methods include the following:C_(max), T_(max), t½, area under the serum concentration time curve fromtime zero to the last measurable concentration (AUC_(0-t)), AUC fromtime zero to infinity (AUC_(0-∞)), clearance (CL), and volume ofdistribution (V_(ss), V_(z)). All PK parameters, including exposure(C_(max), AUCs) to Hu-5F9-G4 will be summarized for individual patientsand by dose cohorts. Exploratory analyses may be conducted to assess therelationship between one or more PK parameters and selected safety andefficacy measures (for example, hemoglobin, reticulocytosis, receptorsaturation by flow cytometry, or immunogenicity).

Immunogenicity Assessments

The rate and magnitude of anti-Hu5F9-G4 antibody positivity will beevaluated for individual patients, for each Part A, B, C/dose level, andfor the pooled patient population. Exploratory evaluations may beconducted to determine the relationship between immunogenicity assaypositivity and one or more safety, PK, or efficacy parameters (forexample, drug clearance, AEs, tumor response).

Anti-Tumor Activity

Analysis of tumor response will be conducted for the Evaluable for TumorAssessment Set. The RECIST v 1.1 or the IWG criteria will be applied,and assessment will be per the investigator. The proportion of patientswith CR, PR, SD, stable disease sustained for 6 months (SD6), and PDwill be calculated at each time point. Objective response will becalculated as the CR+PR with the 95% confidence intervals for each PartA, B, C/dose level and overall for each measurement time point will betabulated. The proportion of patients defined as achieving clinicalbenefit will be calculated as the CR+PR+SD6 with the 95% confidenceintervals. Best Overall Response will also be evaluated. Duration ofresponse will be calculated from the time that initial response wasfirst identified until the development of PD. Progression is assessedrelative to the smallest tumor measurement. Details with regard to theanalysis of antitumor activity will be specified in the SAP.

In summary, based on the results from the toxicology studies, thenonclinical safety assessment program supports the administration ofHu5F9-G4 (e.g., as an IV infusion) for a clinical trial.

Example 4

In preclinical trials, a serum level of greater than 100 μg/mg has beenshown to be therapeutically effective. The data provided in FIG. 9demonstrates a dose in human patients that provides for this level ofdrug.

Shown in FIG. 9 , each of the graphs represents a dosing cohort of humanpatients with solid tumors, treated with 1 mg/kg priming dose of 5F9 andthe indicated maintenance doses. Each line shows the mean value of theconcentration of free drug (Hu5F9-G4) in μg/ml in a serum sample, witherror bars for 3 patients. The values in the table below show the meanCmax for each cohort at week 2 and the AUC last shows how long thesehave been maintained (hours).

The X axis of the graphs depicts the time of samples with respect todosing, where 0 indicates the sample prior to infusion and the remainingare time points by hours post-infusion.

The line marked Week 2 depicts the concentration curve after the firstmaintenance dose at 3, 10, or 20 mg/ml (week one is the priming dose).The data for Week 5 represents the values after the fourth maintenancedose. The highest point of each curve determines the Cmax, and the slopecurve determines the clearance or sustainment of the drug in the serum.

The data from a first maintenance dose of 3 mg/kg shows a rapidclearance from serum. This dose was also cleared fairly quickly evenafter week 5 of treatment. It may also be noted that a dose of 3 mg/kgdoes not achieve the targeted serum level of greater than 100 μg/ml,even as Cmax.

At a dose of 10 mg/kg a Cmax at/above 100 μg/ml was achieved, but thelevel was not sustained but it is not well sustained above 100 μg/mlwith the clearance seen after the week 2 dose. The 10 mg/kg dose doesprovide for a more sustained level at week 5, which may be attributed tosaturation of the CD47 sink with the repeated dosing schedule.

At a maintenance dose of 20 mg/kg, sustained serum levels above thetargeted level of 100 μg/ml could be achieved with the first maintenancedose (week 2). As shown in FIG. 9 , the curve is flat—almost horizontal.

1-21. (canceled)
 22. A pharmaceutical aqueous formulation comprising aneffective dose of Hu5F9-G4 antibody; and pharmaceutically acceptableexcipients comprising 10 mM sodium Acetate, 5% (w/v) sorbitol, 0.01%(w/v) polysorbate 20, pH 5.0.
 23. The formulation of claim 22, whereinthe effective dose of Hu5F9-G4 antibody comprises from 10 to 40 mg/kg.24. The formulation of claim 22, comprising 200 mg of Hu5F9-G4 antibody.25. The formulation of claim 22 in a liquid dosage form for IV infusion.26. The formulation of claim 22, in single use 10 ml. vials.
 27. Anarticle of manufacture comprising a unit dose of a formulation accordingto claim
 22. 28. A method of treating an individual in need thereof byadministering an effective dose of an antibody formulation according toclaim 22.